Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

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Diagnostic Microbiology and Infectious Disease

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Molecular characterization of Klebsiella pneumoniae isolated from renaltransplanted patients: virulence markers, extended-spectrumβ-lactamases, and genetic relatedness☆

Vera Calhau a,b, Luísa Boaventura b, Graça Ribeiro b, Nuno Mendonça a, Gabriela J. da Silva a,⁎a Center of Pharmaceutical Studies and Laboratory of Microbiology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugalb Service of Clinical Pathology, University Hospital of Coimbra, Coimbra, Portugal

a b s t r a c ta r t i c l e i n f o

☆ The results of this work were partially presented at tClinical Microbiology and Infectious Diseases (ECCMID 2⁎ Corresponding author. Tel.: +351-239488460.

E-mail addresses: gjsilva@ci.uc.pt, silva.gj@gmail.com

http://dx.doi.org/10.1016/j.diagmicrobio.2013.08.0310732-8893/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Calhau V, et al,virulence markers, extended-spectru..., Dia

Article history:Received 7 April 2013Received in revised form 9 July 2013Accepted 29 August 2013Available online xxxx

Keywords:Pathogenicity islandPlasmid replicon typingCTX-M-15Virulence factors

The objective was to characterize virulence markers and β-lactam resistance in Klebsiella pneumoniae isolatesfrom renal transplant patients and to evaluate their genetic relatedness. Two main genetic lineageswere detected: 1 carried blaCTX-M-15 not associated to IncFIIA plasmid replicon, which was found on the otherlineage not expressing CTX-M–type enzyme. PAI III536 and PAI IICFT073 were detected for the first timein K. pneumoniae in 1 clone, while the siderophore kfu was carried by the other, with only PAI IV536.The molecular data indicate colonization before admission and fuel the discussion on implementation ofantibiotherapy before surgery.

he 22nd European Congress of012).

(G.J. da Silva).

Molecular characterization of Klebsiella pneugn Microbiol Infect Dis (2014), http://dx.doi.

© 2014 Elsevier Inc. All rights reserved.

Urinary tract infection constitutes the most common infectionamong renal transplant patients. Klebsiella pneumoniae has beenincreasingly isolated, and it has assumed clinical relevance in somehospitals due to the emergence of multidrug-resistant strains. Antimi-crobial resistance limits therapeutic options, increasing the rates oftreatment failure and infection-related mortality, also extendinghospitalization and health care–related costs. K. pneumoniae strainsproducing extended-spectrum β-lactamases (ESBLs) have been report-ed in renal transplant units. However, in contrast with antimicrobialresistance, little is known about the virulence traits of K. pneumoniaestrains isolated from these particular patients. For example, pathoge-nicity islands (PAIs) have been extensively described in urinarypathogenic Escherichia coli (Sabaté et al., 2006), but to our knowledge,only PAI IV536 has been identified so far in K. pneumoniae.

In 2011, an outbreak of multidrug-resistant K. pneumoniae isolateswas identified at the university hospital among renal transplantpatients, raising the hypothesis of an emergent clone and its possibledissemination among wards. The goal of this study was to evaluatethe clonal relatedness of these isolates and to characterize atmolecular level the virulence features and antimicrobial resistanceof the isolates.

From May until July of 2011, 25 K. pneumoniae isolates werecollected from 22 inpatients submitted to a renal transplant at

Coimbra University Hospital, a tertiary care 1200-bed hospital.Identification and susceptibility testing was performed by usingVitek 2 System (BioMérieux, Marcy l’Étoile, France). ESBL productionwas additionally confirmed by using the double disk synergy test. Thegenetic relatedness of K. pneumoniae isolates was analyzed by pulsed-field gel electrophoresis (PFGE), as previously described (Mendonçaet al., 2009). A cutoff value of 80% similarity was determined by thecluster cutoff method according to Bionumerics software. Isolateswith a Dice band-based similarity coefficient value N80% wereconsidered to belong to the same clone. The blaCTX-M, blaSHV, and blaTEMdeterminants were screened and sequenced at Stabvida, Portugal. PAIsmarkers were screened using the method of Bronowski et al. (2008).Other virulence factors such as allS; rmpA gene; the capsular antigengenes K1, K2, K5, K20, K54, and K57; adhesin genes fimH coding for type1 fimbriae, mrkD coding for type 3 fimbriae, and cf29A coding fornonfimbrial adhesin CF29K; and kfu, uge,wabG, and ureawere detectedas described elsewhere (Brisse et al., 2009; Turton et al., 2010). Plasmidreplicon typing was performed using a PCR-based scheme described byCarattoli et al. (2005).

The majority of isolates were collected from urine (76%), 12%from blood, and 12% from exsudates. All the isolates were resistant toampicillin and cefotaxime. High frequency levels of resistance werealso observed for ceftazidime (96%), ciprofloxacin (96%), levofloxacin(96%), trimethoprim/sulfamethoxazole (96%), and gentamicin (88%).They were susceptible to meropenem and ertapenem. KPC enzymeshave already been found in Lisbon, Portugal (Machado et al., 2010).Isolates presented the usual blaSHV gene, and 68% produced CTX-M-15and TEM-1b β-lactamases. The plasmid replicon Inc FIIA was the

moniae isolated from renal transplanted patients:org/10.1016/j.diagmicrobio.2013.08.031

Table 1Antimicrobial resistance and virulence determinants of the main genetic clusters.

Cluster Resistanceprofile

Replicontyping

β-Lactamasesgenes

Virulence markers

I AMP, CTX,CAZ,CIP, LEV, GEN, SXT

ND blaCTX-M-15,blaTEM-1b,blaSHV-type

PAI IV536, fimH, mrkD,kfu, uge, wabG, ureA

II AMP, CTX,CAZ,CIP, LEV, GEN, SXT

ND blaCTX-M-15,blaTEM-1b,blaSHV-type

PAI IV536, fimH, mrkD,kfu, uge, wabG, ureA

III AMP, CTX,CAZ,CIP, LEV, GEN, SXT

FIIA blaSHV-type PAI III536, PAI IICFT073,fimH, mrkD, uge,wabG, ureA

IV AMP, CTX,CAZ,CIP, LEV, GEN, SXT

FIIA blaSHV-type PAI III536, PAI IICFT073,fimH, mrkD, uge,wabG, ureA

Abbreviations: AMP = ampicillin; CTX = cefotaxime; CAZ = ceftazidime; LEV =levofloxacin;GEN=gentamycin; SXT=trimethprim-sulphametoxazol;ND=not detected.The capsular genes K1, K2, K5, K20, K54, and K57; virulence genes alls, rmpA, and cf29Aand PAIs PAI I536, PAI II536, PAI IJ96, PAI IIJ96, and PAI ICFT073 were not detected.

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unique type identified, detected in 32% of the isolates. In Portugal,previous studies reported the presence of blaCTX-M-15 in K. pneumoniae(Conceição et al., 2005; Mendonça et al., 2009), but to our knowledge,this is the first study made in renal transplant patients. IncFII,IncA/C, IncL/M, IncL1 plasmids are frequently found in enterobac-teria and associated with ESBL dissemination (Carattoli, 2011).However, we could not associate FIIA replicon type with CTX-M-15gene. This suggests the presence of other plasmids or a chromo-somal location and that some lineages of K. pneumoniae areevolving to maintain specific antimicrobial determinants. Recently,CTX-M-15 gene was found integrated in K. pneumoniae chromo-some (Coelho et al., 2010).

So far, the extent of knowledge of the virulence traits contentin K. pneumoniae compared to uropathogenic E. coli is limited. Thebiological basis of the clone lineages for divergent clinical behavioris unclear. However, it likely involves great differences in disease-relevant traits, i.e., specific virulence factors. PAI IV536 was foundin 64% of the isolates. PAI III536 and PAI IICFT073 were detectedsimultaneously in 36% of the isolates. PAI I536, PAI II536, PAI IJ96, PAIIIJ96, and PAI ICFT073 were not detected. PAIs arewell described in E. coliuropathogenic strains (Bronowski et al., 2008; Sabaté et al., 2006).However, the presence of these clusters of virulence genes is not sowell known in Klebsiella spp., including in isolates associated withurinary infections. To our knowledge, with the exception of PAI IV536,(Koczura and Kaznowski, 2003), the presence of PAIs in K. pneumoniaestrains has not been investigated.

The virulence encoding-genes fimH, mrkD, uge, wabG, and ureAwere detected in all the isolates, while kfu was detected in 64% of theisolates. alls, rmpA, and cf29A genes and the capsular genes K1, K2, K5,K20, K54, and K57 were not detected.

The analysis of PFGE data revealed the identification of 4 clustersat N80% degree of genetic similarity (Fig. 1). Five isolates did not groupat this cutoff. Clusters I and II were related at a 72% degreeof similarity, while clusters III and IV grouped together at 76%.Table 1 shows the antimicrobial profiles of the main clusters withthe β-lactamase resistance determinants and the virulence genesidentified. The isolates seemed to have distinct origins. Indeed, somepatients could be colonized before admission to the hospital, sincethey were submitted to hemodialysis elsewhere or they weretransferred from regional hospitals. Thus, the screen for colonizationand the knowledge of clinical history on the admission to the tertiary

Fig. 1. Dendrogram based on the band-based coefficient of similarity of PFGE p

Please cite this article as: Calhau V, et al, Molecular characterization ovirulence markers, extended-spectru..., Diagn Microbiol Infect Dis (201

care hospital may be helpful to prevent andmanage further infections.Moreover, the emergence of multidrug-resistant strains in renaltransplant recipients fuels the discussion on whether antibiotherapyshould be implemented before the transplant. Molecular character-ization of bacterial populations can give insight on the sources andmodes of transmission of microorganisms, which is useful to improveor design infection control strategies.

Acknowledgments

All authors contributed equally to this work. N. Mendonça wassupported by a grant SFRH/BPD/45815/2008 from Fundação paraa Ciência e a Tecnologia, Lisbon, Portugal. This work was supportedfinancially by the European Society of Clinical Microbiology andInfectious Diseases 2010 Research Grant and by the Center forPharmaceutical Studies, University of Coimbra, Portugal. The resultsof this work were partially presented at ECCMID 2012.

The positive controls for the plasmid replicon typing were gentlyprovided by Dr Alessandra Carattoli, Instituto Superiore di Sanità,Rome, Italy.

rofiles. Coefficient value N80% was considered to belong to the same clone.

f Klebsiella pneumoniae isolated from renal transplanted patients:4), http://dx.doi.org/10.1016/j.diagmicrobio.2013.08.031

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The authors would like to thank to the members of the BacteriologyLaboratory of the Clinical Pathology Service of University Hospitals ofCoimbra for the collaboration in isolation and identification of bacteriaand to Applied Maths (Belgium) for providing the Bionumerics v6.6software used in this study.

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